Transcriptome analyses of the Giardia lamblia life cycle

Abstract

We quantified mRNA abundance from 10 stages in the Giardia lamblia life cycle in vitro using Serial Analysis of Gene Expression (SAGE). 163 abundant transcripts were expressed constitutively. 71 transcripts were upregulated specifically during excystation and 42 during encystation. Nonetheless, the transcriptomes of cysts and trophozoites showed major differences. SAGE detected co-expressed clusters of 284 transcripts differentially expressed in cysts and excyzoites and 287 transcripts in vegetative trophozoites and encysting cells. All clusters included known genes and pathways as well as proteins unique to Giardia or diplomonads. SAGE analysis of the Giardia life cycle identified a number of kinases, phosphatases, and DNA replication proteins involved in excystation and encystation, which could be important for examining the roles of cell signaling in giardial differentiation. Overall, these data pave the way for directed gene discovery and a better understanding of the biology of G. lamblia.

Figure 1

A) Hierarchical cluster analysis of 697 differentially expressed (R ≥ 8) transcripts from 10 stages in the complete G. lamblia life cycle. Red indicates upregulation and green indicates downregulation relative to the median abundance (black) for each transcript. Hierarchical clustering was performed with all transcript SAGE frequencies log transformed, median centering for both transcripts and SAGE libraries, and clustering of both using centered correlation and the average linkage clustering method. Δ denotes a sub-cluster of 4 transcripts upregulated during both encystation and excystation (“Differentiation” in Table S1). ⇓ denotes a cluster of 9 transcripts downregulated in cysts and S1 (“Down Cyst, S1” in Table S1). B) Relative abundance of differentially expressed transcripts (sense only) presented as histograms color-coded according to the functional categories of their encoded proteins (see Fig. 2).

Figure 2

Key for functional categories of differentially expressed (R ≥ 8) Giardia proteins (see Table S1). Functional categories were assigned by searching UniProtKB/Swiss-Prot with BLASTP (e-values ≤ e⁻⁶) using WU-Blast2 and labeling the positive hits with the identified Swiss-Prot functional category, as well as prediction of functional domains using the Pfam profile HMM database. Included in the classification are categories for proteins unique to Giardia (no homologs detected in GenBank) or within the diplomonads (based on comparison to available Spironucleus vortens EST data), as well as for proteins with homology to hypothetical proteins found in other genomes (“Conserved Hypothetical” in Table S1).